Photocontrol of the ß-Hairpin Polypeptide Structure through an Optimized Azobenzene-Based Amino Acid Analogue.

A family of neurodegenerative diseases, including Huntington's disease (HD) and spinocerebellar ataxias, are associated with an abnormal polyglutamine (polyQ) expansion in mutant proteins that become prone to form amyloid-like aggregates. Prior studies have suggested a key role for ß-hairpin formation as a driver of nucleation and aggregation, but direct experimental studies have been challenging. Toward such research, we set out to enable spatiotemporal control over ß-hairpin formation by the introduction of a photosensitive ß-turn mimic in the polypeptide backbone, consisting of a newly designed azobenzene derivative. The reported derivative overcomes the limitations of prior approaches associated with poor photochemical properties and imperfect structural compatibility with the desired ß-turn structure. A new azobenzene-based ß-turn mimic was designed, synthesized, and found to display improved photochemical properties, both prior and after incorporation into the backbone of a polyQ polypeptide. The two isomers of the azobenzene-polyQ peptide showed different aggregate structures of the polyQ peptide fibrils, as demonstrated by electron microscopy and solid-state NMR (ssNMR). Notably, only peptides in which the ß-turn structure was stabilized (azobenzene in the cis configuration) closely reproduced the spectral fingerprints of toxic, ß-hairpin-containing fibrils formed by mutant huntingtin protein fragments implicated in HD. These approaches and findings will enable better deciphering of the roles of ß-hairpin structures in protein aggregation processes in HD and other amyloid-related neurodegenerative diseases.

Solid-state nuclear magnetic resonance in the structural study of polyglutamine aggregation.

The aggregation of proteins into amyloid-like fibrils is seen in many neurodegenerative diseases. Recent years have seen much progress in our understanding of these misfolded protein inclusions, thanks to advances in techniques such as solid-state nuclear magnetic resonance (ssNMR) spectroscopy and cryogenic electron microscopy (cryo-EM). However, multiple repeat-expansion-related disorders have presented special challenges to structural elucidation. This review discusses the special role of ssNMR analysis in the study of protein aggregates associated with CAG repeat expansion disorders. In these diseases, the misfolding and aggregation affect mutant proteins with expanded polyglutamine segments. The most common disorder, Huntington's disease (HD), is connected to the mutation of the huntingtin protein. Since the discovery of the genetic causes for HD in the 1990s, steady progress in our understanding of the role of protein aggregation has depended on the integrative and interdisciplinary use of multiple types of structural techniques. The heterogeneous and dynamic features of polyQ protein fibrils, and in particular those formed by huntingtin N-terminal fragments, have made these aggregates into challenging targets for structural analysis. ssNMR has offered unique insights into many aspects of these amyloid-like aggregates. These include the atomic-level structure of the polyglutamine core, but also measurements of dynamics and solvent accessibility of the non-core flanking domains of these fibrils' fuzzy coats. The obtained structural insights shed new light on pathogenic mechanisms behind this and other protein misfolding diseases.

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission.

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission.

Fast magic angle spinning for the characterization of milligram quantities of organic and biological solids at natural isotopic abundance by 13C-13C correlation DNP-enhanced NMR.

We show that multidimensional solid-state NMR 13C-13C correlation spectra of biomolecular assemblies and microcrystalline organic molecules can be acquired at natural isotopic abundance with only milligram quantities of sample. These experiments combine fast Magic Angle Spinning of the sample, low-power dipolar recoupling, and dynamic nuclear polarization performed with AsymPol biradicals, a recently introduced family of polarizing agents. Such experiments are essential for structural characterization as they provide short- and long-range distance information. This approach is demonstrated on diverse sample types, including polyglutamine fibrils implicated in Huntington's disease and microcrystalline ampicillin, a small antibiotic molecule.

Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation.

Apolipoprotein A-I (apoA-I) shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and intermolecularly, providing structural stabilization by self-association. We have reported that lipid-free apoA-I becomes amyloidogenic upon physiologically relevant (myeloperoxidase-mediated) Met oxidation. In this study, we established that Met oxidation promotes amyloidogenesis by reducing the stability of apoA-I monomers and irreversibly disrupting self-association. The oxidized apoA-I monomers also exhibited increased cellular cholesterol release capacity and stronger association with macrophages, compared to nonoxidized apoA-I. Of physiologic relevance, preformed oxidized apoA-I amyloid fibrils induced amyloid formation in nonoxidized apoA-I. This process was enhanced when self-association of nonoxidized apoA-I was disrupted by thermal treatment. Solid state NMR analysis revealed that aggregates formed by seeded nonoxidized apoA-I were structurally similar to those formed by the oxidized protein, featuring a ß-structure-rich amyloid fold alongside α-helices retained from the native state. In atherosclerotic lesions, the conditions that promote apoA-I amyloid formation are readily available: myeloperoxidase, active oxygen species, low pH, and high concentration of lipid-free apoA-I. Our results suggest that even partial Met oxidation of apoA-I can nucleate amyloidogenesis, thus sequestering and inactivating otherwise antiatherogenic and HDL-forming apoA-I.-Witkowski, A., Chan, G. K. L., Boatz, J. C., Li, N. J., Inoue, A. P., Wong, J. C., van der Wel, P. C. A., Cavigiolio, G. Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation.

Hidden motions and motion-induced invisibility: Dynamics-based spectral editing in solid-state NMR.

Solid-state nuclear magnetic resonance (ssNMR) spectroscopy enables the structural characterization of a diverse array of biological assemblies that include amyloid fibrils, non-amyloid aggregates, membrane-associated proteins and viral capsids. Such biological samples feature functionally relevant molecular dynamics, which often affect different parts of the sample in different ways. Solid-state NMR experiments' sensitivity to dynamics represents a double-edged sword. On the one hand, it offers a chance to measure dynamics in great detail. On the other hand, certain types of motion lead to signal loss and experimental inefficiencies that at first glance interfere with the application of ssNMR to overly dynamic proteins. Dynamics-based spectral editing (DYSE) ssNMR methods leverage motion-dependent signal losses to simplify spectra and enable the study of sub-structures with particular motional properties.

Huntingtin exon 1 fibrils feature an interdigitated ß-hairpin-based polyglutamine core.

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of ß-hairpin-containing ß-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical ß-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

Structural Fingerprinting of Protein Aggregates by Dynamic Nuclear Polarization-Enhanced Solid-State NMR at Natural Isotopic Abundance.

A pathological hallmark of Huntington's disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13C-13C distance constraints.

On the use of ultracentrifugal devices for routine sample preparation in biomolecular magic-angle-spinning NMR.

A number of recent advances in the field of magic-angle-spinning (MAS) solid-state NMR have enabled its application to a range of biological systems of ever increasing complexity. To retain biological relevance, these samples are increasingly studied in a hydrated state. At the same time, experimental feasibility requires the sample preparation process to attain a high sample concentration within the final MAS rotor. We discuss these considerations, and how they have led to a number of different approaches to MAS NMR sample preparation. We describe our experience of how custom-made (or commercially available) ultracentrifugal devices can facilitate a simple, fast and reliable sample preparation process. A number of groups have since adopted such tools, in some cases to prepare samples for sedimentation-style MAS NMR experiments. Here we argue for a more widespread adoption of their use for routine MAS NMR sample preparation.

Insights into protein misfolding and aggregation enabled by solid-state NMR spectroscopy.

The aggregation of proteins and peptides into a variety of insoluble, and often non-native, aggregated states plays a central role in many devastating diseases. Analogous processes undermine the efficacy of polypeptide-based biological pharmaceuticals, but are also being leveraged in the design of biologically inspired self-assembling materials. This Trends article surveys the essential contributions made by recent solid-state NMR (ssNMR) studies to our understanding of the structural features of polypeptide aggregates, and how such findings are informing our thinking about the molecular mechanisms of misfolding and aggregation. A central focus is on disease-related amyloid fibrils and oligomers involved in neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's disease. SSNMR-enabled structural and dynamics-based findings are surveyed, along with a number of resulting emerging themes that appear common to different amyloidogenic proteins, such as their compact alternating short-ß-strand/ß-arc amyloid core architecture. Concepts, methods, future prospects and challenges are discussed.

MAS 1H NMR Probes Freezing Point Depression of Water and Liquid-Gel Phase Transitions in Liposomes.

The lipid bilayer typical of hydrated biological membranes is characterized by a liquid-crystalline, highly dynamic state. Upon cooling or dehydration, these membranes undergo a cooperative transition to a rigidified, more-ordered, gel phase. This characteristic phase transition is of significant biological and biophysical interest, for instance in studies of freezing-tolerant organisms. Magic-angle-spinning (MAS) solid-state NMR (ssNMR) spectroscopy allows for the detection and characterization of the phase transitions over a wide temperature range. In this study we employ MAS 1H NMR to probe the phase transitions of both solvent molecules and different hydrated phospholipids, including tetraoleoyl cardiolipin (TOCL) and several phosphatidylcholine lipid species. The employed MAS NMR sample conditions cause a previously noted substantial reduction in the freezing point of the solvent phase. The effect on the solvent is caused by confinement of the aqueous solvent in the small and densely packed MAS NMR samples. In this study we report and examine how the freezing point depression also impacts the lipid phase transition, causing a ssNMR-observed reduction in the lipids' melting temperature (Tm). The molecular underpinnings of this phenomenon are discussed and compared with previous studies of membrane-associated water phases and the impact of membrane-protective cryoprotectants.

Peptide-Directed Assembly of Single-Helical Gold Nanoparticle Superstructures Exhibiting Intense Chiroptical Activity.

Chiral nanoparticle assemblies are an interesting class of materials whose chiroptical properties make them attractive for a variety of applications. Here, C18-(PEPAuM-ox)2 (PEPAuM-ox = AYSSGAPPMoxPPF) is shown to direct the assembly of single-helical gold nanoparticle superstructures that exhibit exceptionally strong chiroptical activity at the plasmon frequency with absolute g-factor values up to 0.04. Transmission electron microscopy (TEM) and cryogenic electron tomography (cryo-ET) results indicate that the single helices have a periodic pitch of approximately 100 nm and consist of oblong gold nanoparticles. The morphology and assembled structure of C18-(PEPAuM-ox)2 are studied using TEM, atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, X-ray diffraction (XRD), and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. TEM and AFM reveal that C18-(PEPAuM-ox)2 assembles into linear amyloid-like 1D helical ribbons having structural parameters that correlate to those of the single-helical gold nanoparticle superstructures. FTIR, CD, XRD, and ssNMR indicate the presence of cross-ß and polyproline II secondary structures. A molecular assembly model is presented that takes into account all experimental observations and that supports the single-helical nanoparticle assembly architecture. This model provides the basis for the design of future nanoparticle assemblies having programmable structures and properties.

Structural Changes and Proapoptotic Peroxidase Activity of Cardiolipin-Bound Mitochondrial Cytochrome c.

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly (13)C,(15)N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.

Polyglutamine amyloid core boundaries and flanking domain dynamics in huntingtin fragment fibrils determined by solid-state nuclear magnetic resonance.

In Huntington's disease, expansion of a polyglutamine (polyQ) domain in the huntingtin (htt) protein leads to misfolding and aggregation. There is much interest in the molecular features that distinguish monomeric, oligomeric, and fibrillar species that populate the aggregation pathway and likely differ in cytotoxicity. The mechanism and rate of aggregation are greatly affected by the domains flanking the polyQ segment within exon 1 of htt. A "protective" C-terminal proline-rich flanking domain inhibits aggregation by inducing polyproline II structure (PPII) within an extended portion of polyQ. The N-terminal flanking segment (htt(NT)) adopts an α-helical structure as it drives aggregation, helps stabilize oligomers and fibrils, and is seemingly integral to their supramolecular assembly. Via solid-state nuclear magnetic resonance (ssNMR), we probe how, in the mature fibrils, the htt flanking domains impact the polyQ domain and in particular the localization of the ß-structured amyloid core. Using residue-specific and uniformly labeled samples, we find that the amyloid core occupies most of the polyQ domain but ends just prior to the prolines. We probe the structural and dynamical features of the remarkably abrupt ß-sheet to PPII transition and discuss the potential connections to certain htt-binding proteins. We also examine the htt(NT) α-helix outside the polyQ amyloid core. Despite its presumed structural and demonstrated stabilizing roles in the fibrils, quantitative ssNMR measurements of residue-specific dynamics show that it undergoes distinct solvent-coupled motion. This dynamical feature seems reminiscent of molten-globule-like α-helix-rich features attributed to the nonfibrillar oligomeric species of various amyloidogenic proteins.

Combinations of arginine and pullulan reveal the selective effect of stabilization mechanisms on different lyophilized proteins.

The stability of lactate dehydrogenase (LDH) and ß-galactosidase (ß-gal), incorporated in arginine/pullulan (A/P) mixtures at various weight ratios by lyophilization, was determined. The physicochemical characteristics of various A/P mixtures were assessed. With decreasing A/P ratios, the glass transition temperature of the formulations increased. Furthermore, arginine crystallization due to high relative humidity (RH) exposure was prevented at an A/P weight ratio of 4/6 or less. When stored at 0 % RH / 60 °C for 4 weeks, arginine was superior to pullulan as stabilizer. During storage at 43 % RH / 30 ℃ for 4 weeks, the enzymatic activity of LDH was best retained at an A/P weight ratio of 2/8, while ß-gal activity was relatively well-retained at A/P weight ratios of both 8/2 and 2/8. LDH seemed to be more prone to degradation in the rubbery state. In the glassy state, ß-gal degraded faster than LDH. Solid-state nuclear magnetic resonance spectroscopy showed that (labeled) arginine experienced a different interaction in the two protein samples, reflecting a modulation of long-range correlations of the arginine side chain nitrogen atoms (Nε, Nη). In summary, LDH stabilization in the A/P matrix requires vitrification. Further stabilization difference between LDH and ß-gal may be dependent on the interaction with arginine.

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR.

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

Integrative determination of the atomic structure of mutant huntingtin exon 1 fibrils from Huntington's disease.

Neurodegeneration in Huntington's disease (HD) is accompanied by the aggregation of fragments of the mutant huntingtin protein, a biomarker of disease progression. A particular pathogenic role has been attributed to the aggregation-prone huntingtin exon 1 (HttEx1) fragment, whose polyglutamine (polyQ) segment is expanded. Unlike amyloid fibrils from Parkinson's and Alzheimer's diseases, the atomic-level structure of HttEx1 fibrils has remained unknown, limiting diagnostic and treatment efforts. We present and analyze the structure of fibrils formed by polyQ peptides and polyQ-expanded HttEx1. Atomic-resolution perspectives are enabled by an integrative analysis and unrestrained all-atom molecular dynamics (MD) simulations incorporating experimental data from electron microscopy (EM), solid-state NMR, and other techniques. Visualizing the HttEx1 subdomains in atomic detail helps explaining the biological properties of these protein aggregates, as well as paves the way for targeting them for detection and degradation.

New insights in polydopamine formation via surface adsorption.

Polydopamine is a biomimetic self-adherent polymer, which can be easily deposited on a wide variety of materials. Despite the rapidly increasing interest in polydopamine-based coatings, the polymerization mechanism and the key intermediate species formed during the deposition process are still controversial. Herein, we report a systematic investigation of polydopamine formation on halloysite nanotubes; the negative charge and high surface area of halloysite nanotubes favour the capture of intermediates that are involved in polydopamine formation and decelerate the kinetics of the process, to unravel the various polymerization steps. Data from X-ray photoelectron and solid-state nuclear magnetic resonance spectroscopies demonstrate that in the initial stage of polydopamine deposition, oxidative coupling reaction of the dopaminechrome molecules is the main reaction pathway that leads to formation of polycatecholamine oligomers as an intermediate and the post cyclization of the linear oligomers occurs subsequently. Furthermore, TRIS molecules are incorporated into the initially formed oligomers.

Anomalous peroxidase activity of cytochrome c is the primary pathogenic target in Barth syndrome.

Barth syndrome (BTHS) is a life-threatening genetic disorder with unknown pathogenicity caused by mutations in TAFAZZIN (TAZ) that affect remodeling of mitochondrial cardiolipin (CL). TAZ deficiency leads to accumulation of mono-lyso-CL (MLCL), which forms a peroxidase complex with cytochrome c (cyt c) capable of oxidizing polyunsaturated fatty acid-containing lipids. We hypothesized that accumulation of MLCL facilitates formation of anomalous MLCL-cyt c peroxidase complexes and peroxidation of polyunsaturated fatty acid phospholipids as the primary BTHS pathogenic mechanism. Using genetic, biochemical/biophysical, redox lipidomic and computational approaches, we reveal mechanisms of peroxidase-competent MLCL-cyt c complexation and increased phospholipid peroxidation in different TAZ-deficient cells and animal models and in pre-transplant biopsies from hearts of patients with BTHS. A specific mitochondria-targeted anti-peroxidase agent inhibited MLCL-cyt c peroxidase activity, prevented phospholipid peroxidation, improved mitochondrial respiration of TAZ-deficient C2C12 myoblasts and restored exercise endurance in a BTHS Drosophila model. Targeting MLCL-cyt c peroxidase offers therapeutic approaches to BTHS treatment.

Structural characterization of the caveolin scaffolding domain in association with cholesterol-rich membranes.

Members of the caveolin protein family are implicated in the formation of caveolae and play important roles in a number of signaling pathways and in the regulation of various proteins. We employ complementary spectroscopic methods to study the structure of the caveolin scaffolding domain (CSD) in caveolin-1 fragments, while bound to cholesterol-rich membranes. This key domain is thought to be involved in multiple critical functions that include protein recognition, oligomerization, and cholesterol binding. In our membrane-bound peptides, residues within the flanking intramembrane domain (IMD) are found to adopt an α-helical structure, consistent with its commonly believed helical hairpin conformation. Intriguingly, in these same peptides, we observe a ß-stranded conformation for residues in the CSD, contrasting with earlier reports, which commonly do not reflect ß-structure. Our experimental data based on solid-state NMR, CD, and FTIR are found to be consistent with computational analyses of the secondary structure preference of the primary sequence. We discuss how our structural data of membrane binding Cav fragments may match certain general features of cholesterol-binding domains and could be consistent with the role for CSD in protein recognition and homo-oligomerization.